This Application is a 35 USC 371 of PCT/GB97/02523, filed Sep. 11, 1997.
1. Field of the Invention
The present invention relates to materials and methods for the diagnosis and treatment of pre-eclampsia and diabetes, and more particularly to the role of P-type inositolphosphoglycans (IPGs) in the occurrence of pre-eclampsia, methods of diagnosing pre-eclampsia and uses of antagonists of P-type IPGs in the treatment of pre-eclampsia.
2. Background of the Invention
Pre-eclampsia is a placental disease [1] characterised by insufficiency of the uteroplacental circulation [2], and which affects 10-12% of all pregnancies and is a major factor in the perinatal mortality rate. There is evidence that one or more placentally-derived factors are released into the maternal circulation which either directly or indirectly cause maternal endothelial dysfunction and ensuing maternal problems with activation of the clotting system increased vascular permeability and ischaemia in maternal organs secondary to vasoconstriction [3].
The present invention arises from investigations to determine whether there is a correlation between pre-eclampsia and its degree of severity and the profile of inositol phosphoglycans (IPGs) in the pre-eclamptic subjects and their normal age and parity-matched controls. In order to gain information on the significance of the disordered carbohydrate metabolism in the placenta in pre-eclampsia, as previously revealed by the massive increase in glycogen accumulation [4], comparison has been made with diabetic pregnant women in which placental glycogen accumulation is also a prominent feature [4,5], although not accompanied by the same degree of the life-threatening sequelae of pre-eclampsia.
Accordingly, in a first aspect, the present invention provides the use of a P-type inositolphosphoglycan (IPG) antagonist in the preparation of a medicament for the treatment of pre-eclampsia.
In further aspect, the present invention provides a method of treating pre-eclampsia in a patient, the method comprising administering a therapeutically effective amount of a P-type IPG antagonist to a patient.
In a further aspect, the present invention provides a pharmaceutical composition comprising a P-type antagonist in combination with a pharmaceutically acceptable carrier.
P-type IPGs and method for isolating them from human tissue are described below. This in turn allows those of ordinary skill in the art to prepare P-type IPG antagonists.
In the present invention, xe2x80x9cP-type IPG antagonistsxe2x80x9d includes substances which have one or more of the following properties:
(a) inhibiting the release of P-type IPG from placenta;
(b) reducing the levels of placenta derived P-type IPG via an IPG binding substance (e.g. an antibody or a specific binding protein) against the placental derived TPG; and/or,
(c) reducing the effects of placenta derived P-type IPG.
In a further aspect, the present invention provides a method of screening for P-type IPG antagonists, the method comprising:
(a) contacting a candidate antagonist and a P-type IPG in an assay for a biological property of the P-type IPG under conditions in which the P-type IPG and the candidate antagonist can compete;
(b) measuring the biological property of the P-type IPG; and,
(c) selecting candidate antagonists which reduce the biological activity of the P-type IPG.
Some of the biological properties of P-type IPGs and assays to determine these properties that can be used in the above screening method are set out in the description below. The techniques of combinatorial chemistry are particularly suited to the production of large numbers of synthetic candidate antagonists, which can be screened for activity in the above method.
The particular emphasis placed upon the determination of the output of IPGs in both pre-eclamptic and diabetic pregnant women relates to the known fundamental importance of the class of compound in regulation key sites in metabolic pathways, resulting, in different tissues, in the direction of carbohydrates towards oxidation and glycogen synthesis in the case of the IPG P-type, or towards lipogenesis in the case of the IPG A-type; this regulation being both organ and inter-organ related [6, 9].
In copending applications claiming priority from GB-A-9618934.5, we report on the urinary content of IPGs in diabetic patients, from which evidence has been adduced for a critical role of altered inositol phosphoglycan profiles in relation to parameters linked to syndrome X, such as insulin resistance, obesity and high blood pressure. There are similarities between the metabolic changes in pre-eclampsia and syndrome X [10].
The results of our investigations described below indicate the following:
(a) The 24 hour output of IPG P-type in urine in pre-eclamptic women is significantly higher (2- to 3-fold) than in pregnant control subjects matched for age, parity and stage of gestation.
(b) Diabetic pregnant women do not show any significant change in urinary output of IPG P-type relative to pregnant control subjects matched for age, parity and gestational stage.
(c) Pregnancy itself is associated with an increased urinary output of IPG P-type relative to non-pregnant controls matched for age.
(d) No significant changes were found in the daily output of IPG A-type in pre-eclamptic or diabetic groups, with the exception of an increase in the IPG A-type in the pre-eclamptic group when the results were expressed as units per mmole creatinine.
(e) Urinary excretion of IPG P-type correlated with markers of the severity of pre-eclampsia, plasma alanine aspartate transaminase, degree of proteinuria and with platelet counts.
(f) Human placenta contained very high concentrations of IPG-P type, some 100xc3x97greater than either human or rat liver. It also appears to contain an inhibitor of IPG-P-ype activity as evidenced by a calculated decrease in activity when increasing the volume of the same preparation tested in the PDH phosphatase system (FIG. 7). Pre-eclamptic placenta contains approximately twice as much IPG-P type as does placenta from normal pregnant subjects. The IPG-A (pH 1.3 fraction) isolated from placenta showed no activity when tested for its ability to stimulate lipogenesis in rat adipocytes.
(g) Evidence has been found suggesting that the use of contraceptive pills may be related to an increase in IPG P-type in urine of normal women. (5 values only in each group).
(h) After storage for 10 months at xe2x88x928xc2x0 C., urine from pre-eclamptic women showed increased P-type activity (FIG. 8A) indicating that the urine initially contained a labile inhibitor. The yield of the IPG A-type isolated from the same urines decreased in activity (FIG. 8B).
(i) Significant differences were found between the ratios of IPG P-type and IPG A-type in non-pregnant women and normal male subjects; while the IPG P-type was similar in both groups, the IPG A-type was 5- to 6-fold higher in women.
(j) There is a 2.7 fold increase in IPG P-type in the urine of pre-eclamptic women, compared to normal pregnant subjects. There is a 2.7 fold increase in placenta-derived P-type mediators from pre-eclamptic women compared to normal pregnant subjects (See Table 5).
(k) The high urinary excretion IPG P-type in pre-eclampsia reflects high placental and circulating levels and is directly related to the accumulation of glycogen in the placenta in this condition, because IPG P-type activates glycogen synthase phosphatase.
(l) The concentration of P-type mediators in the urine of pre-eclamptic women returns baseline in post natal sample, (See FIG. 6) confirms that the source of the relevant P-type mediator in pre-eclamptic women is the placenta.
(m) A high circulating level of IPG P-type originating in the placenta may have paradrine effects, eg: in stimulating other endocrine glands, and/or affecting endothelial cells which could contribute to the pathogenesis of the pre-eclampsia syndrome.
The present invention provides, inter alia, a therapeutic treatment of pre-eclampsia:
(1) to inhibit the release of P-type mediator from placenta;
(2) to reduce the levels of placenta derived P-type IPG via antibody against the placental derived IPG;
(3) to reduce the effects of placenta derived P-type IPG via P-type antagonist.
The substance can be administered as the sole active substance, or as an adjunct to other forms of treatment. Because of their small molecular weight and heat and acid stability, IPGs should be suitable for oral administration, but other forms of administration are also contemplated. In the case of antibodies, or other proteins or substances which may not be suitable for oral administration, other methods such as parenteral administration may be used. Antibodies for administration are preferably human or xe2x80x9chumanisedxe2x80x9d according to known techniques. This is discussed further below.
The invention also contemplates measurement of P-type IPG in blood or urine as a diagnostic for pre-eclampsia. Thus, in a further aspect, the present invention provides a method of diagnosing pre-eclampsia in a patient, the method comprising determining the level of P-type IPGs in a biological sample: obtained from the patient. Thus, a diagnosis can then be made by correlating this level with known levels of the P-type IPGS.
In one embodiment, the method comprises the steps of:
(a) contacting a biological sample obtained from the patient with a solid support having immobilised thereon binding agent having binding sites specific for one or more P-type IPGs;
(b) contacting the solid support with a labelled developing agent capable of binding to unoccupied binding sites, bound P-type IPGs or occupied binding sites; and,
(c) detecting the label of the developing agent specifically binding in step (b) to obtain a value representative of the level of the P-type IPGs in the sample.
As set out below, in this aspect of the invention, the level of the P-type IPGs can be further confirmed using a marker which correlates with the level of the P-type IPGs.
The present invention will now be described by way of example and not limitation with reference to the accompanying drawings.